title: Co-expression of RNA-protein complexes in Escherichia coli and applications to RNA biology.
abstract: RNA has emerged as a major player in many cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. In this work, we have extended this method to RNA/protein co-expression. We have engineered several plasmids that allow overexpression of RNA-protein complexes in E. coli. We have investigated the potential of these tools in many applications, including the production of nuclease-sensitive RNAs encapsulated in viral protein pseudo-particles, the co-production of non-coding RNAs with chaperone proteins, the incorporation of a post-transcriptional RNA modification by co-production with the appropriate modifying enzyme and finally the production and purification of an RNA-His-tagged protein complex by nickel affinity chromatography. We show that this last application easily provides pure material for crystallographic studies. The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins.
authors.affiliation: CNRS, UMR 8015, Laboratoire de Cristallographie et RMN biologiques, 4 avenue de l'Observatoire, 75006 Paris, France and Université Paris Descartes, Sorbonne Paris Cité, UMR 8015, Laboratoire de Cristallographie et RMN biologiques, 4 avenue de l'Observatoire, 75006 Paris, France.
authors.forename: Luc
authors.initials: L
authors.lastname: Ponchon
authors.forename: Marjorie
authors.initials: M
authors.lastname: Catala
authors.forename: Bili
authors.initials: B
authors.lastname: Seijo
authors.forename: Marguerite
authors.initials: M
authors.lastname: El Khouri
authors.forename: Frédéric
authors.initials: F
authors.lastname: Dardel
authors.forename: Sylvie
authors.initials: S
authors.lastname: Nonin-Lecomte
authors.forename: Carine
authors.initials: C
authors.lastname: Tisné
mesh_terms: D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000378:metabolism; D017909:Levivirus / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000378:metabolism*; D012357:RNA, Transfer, Lys / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D036022:Capsid Proteins; D012329:RNA, Bacterial; D012357:RNA, Transfer, Lys; C039919:RNA, recombinant; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C430328:small protein B; D012313:RNA